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Development and Characterization of a Solid-Phase Biocatalyst Based on Cyclodextrin Glucantransferase Reversibly Immobilized onto Thiolsulfinate-Agarose

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Abstract

Reduction of disulfide bonds and introduction of “de novo” thiol groups in cyclodextrin glucantransferase from Thermoanaerobacter sp. were assessed in order to perform reversible covalent immobilization onto thiol-reactive supports (thiolsulfinate-agarose). Only the thiolation process dramatically improved the immobilization yield, from 0 % for the native and reduced enzyme, up to nearly 90 % for the thiolated enzyme. The mild conditions of the immobilization process (pH 6.8–7.0 and 22 °C) allowed the achievement of 100 % coupling efficiencies when low loads were applied. Ionic strength was a critical parameter for the immobilization process; for high activity recoveries, 50 mM phosphate buffer supplemented with 0.15 M NaCl was required. The kinetic parameters, pH and thermal stabilities for the immobilized biocatalyst were similar to those for the native enzyme. For β-cyclization activity, optimal pH range and temperature were 4.0–5.4 and 85 °C. The possibility of reusing the support was demonstrated by the reversibility of enzyme–support binding.

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Acknowledgements

We are grateful for financial support by the Programa para el Desarrollo de las Ciencias Básicas (PEDECIBA), Uruguay. We also thank NOVOZYMES and its local representative FRADEC for providing free samples of Toruzyme® 3.0 L and Dr. Valerie Dee for linguistic revision.

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  1. Departamento de Biociencias, Cátedra de Bioquímica, Facultad de Química, Universidad de la República, General Flores 2124, CC 1157, Montevideo, Uruguay

    Santiago Einar Viera, Francisco Batista-Viera & Karen Ovsejevi

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  1. Santiago Einar Viera
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  2. Francisco Batista-Viera
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  3. Karen Ovsejevi
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Correspondence to Karen Ovsejevi.

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Viera, S.E., Batista-Viera, F. & Ovsejevi, K. Development and Characterization of a Solid-Phase Biocatalyst Based on Cyclodextrin Glucantransferase Reversibly Immobilized onto Thiolsulfinate-Agarose. Appl Biochem Biotechnol 167, 164–176 (2012). https://doi.org/10.1007/s12010-012-9686-8

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  • DOI: https://doi.org/10.1007/s12010-012-9686-8

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